Protein phosphorylation mediated by protein kinases is the principal mechanism by which eukaryotic cellular processes are modulated by external physiological stimuli. Phosphopeptides are essential tools for the study of this process, serving as model substrates for phosphatases, as antigens for the production of antibodies against phosphorylated proteins, and as reference compounds for determining their physical parameters. The development of methods for the production of phosphopeptides has consequently attracted considerable interest over the last few years, and these endeavours have yielded reliable procedures which have now made their synthesis routine. There are two strategies used currently for the preparation of phosphopeptides: the building block approach, in which pre-formed protected phosphoamino acids are incorporated during the course of chain assembly, and the global phosphorylation method, which involves post-synthetic phosphorylation of serine, threonine, or tyrosine side-chain hydroxyl groups on the solid support. The building block procedure is certainly the more straightforward of the two approaches and has now become, owing to the availability of suitably protected phosphoamino acids, the standard method for the routine production of phosphopeptides. For the side-chain protection of phosphotyrosine in Fmoc/tBu-based solid phase synthesis, methyl, benzyl, t-butyl, dialkylamino, and silyl groups have been employed. Of these, benzyl is most useful as it is the most convenient to introduce and is rapidly removed during the TFA-mediated acidolysis step. Only the mono-benzyl ester, Fmoc-Tyr(PO(OBzl)-OH)-OH 1, is available commercially; the dibenzyl ester offers no practical benefit as it undergoes mono-debenzylation in the course of the piperidine-mediated Fmoc deprotection reaction. Also available commercially is Fmoc-Tyr(PO3H2)-OH 2. This derivative, despite having no phosphate protection, appears to work well, particularly in the synthesis of small- to medium-sized phosphopeptides; although formation of the pyrophosphate 3 can be a problem in peptides containing adjacent Tyr(PO3H2) residues. Phosphate triesters of serine and threonine are not compatible with Fmoc/tBu chemistry as they undergo β-elimination when treated with piperidine, resulting in the formation of dehydroalanine and dehydoaminobutyric acid, respectively For this reason, it was long believed that the building block approach could not be used for preparation of peptides containing these amino acids.
E64 [trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane] analogues as inhibitors of cysteine proteinases: investigation of S2 subsite interactions
